Lecture #14
Regulatory Enzymes
Outline
• Phosphofructokinase-1• Describing the bound states of
activators and inhibitors• Integration with glycolysis
Phosphofructokinase-1
Metabolic Role
Background• Tetramer• 3 Isoforms: M,L,P (muscle, liver, platelet)• 2 Natural Forms: R,T (relaxed, tight)
• Known inhibitors: ATP, citrate, PEP• Known activators: AMP, cAMP, Pi, SO4, FBP• Catalytic Activity:
PFK sub-network
The Catalytic Mechanism:binding of the two substrates followed by the
chemical reaction
1)
2)
3)
AMP and ATP as regulatory ligands
activation
inhibition
conformation
Stoichiometric Matrix
Pools and Ratios• PFK – R state
– All forms of R0 + R1 + R2 + R3 + R4
• PFK – T state– All forms of T0 + T1 + T2 + T3 + T4
• PFK – R catalytic state– All forms of Ri,AF
• Ratios
• At steady state ~ rR = 90%, rcat = 12%
DETERMINING THE STEADY STATE
Let’s revisit the subnetwork
Equilibrium v = 0
Steady State
Constraints on the Network
• Total mass balance:
• Total flux:
• Known equilibrium constants
Solving for the concentrations
Note: When equilibrium constants are plugged in, all forward rate constants in equilibrium reactions fall out, leaving only the catalytic rate constants
Estimating the catalytic rate constants Chosen Steady State
kPFK
kF6P
kATP
INTEGRATION WITH GLYCOLYSIS
Stoichiometric Matrix
DYNAMIC SIMULATIONS
Dynamic Simulation
• Two perturbations– Standard 50% increase in ATP utilization– Additional 15% decrease in ATP utilization
Glycolysis Dynamics
Glycolysis Dynamics
50% increase in ATP utilization 15% decrease in ATP utilization
Summary• Enzymes can be explicitly represented in simulation
modules as molecules• Enzymes have many binding states• Binding of regulators (inhibitors and activators) alters
protein activity; leading to a ‘tug of war’ amongst the functional states (i.e. T and R)
• Ratios that represent what fraction of the enzyme is in an active or inhibited functional states can be formed
• Enzyme sub-networks can be seamlessly integrated with the scaffold metabolic network
• Regulator binding to PFK, a key glycolytic regulatory enzyme, was demonstrated
